Novozym 234 was the most frequently used enzyme for production of Rhizoctonia solani protoplasts. Since manufacture of this enzyme was discontinued in the late 1990s, a new procedure was developed by testing lytic enzymes from Sigma and by examining factors affecting protoplast formation. The combination of 20 mg/mL Driselase and 10 mg/mL lysing enzyme was effective in releasing protoplasts from R. solani. The optimal condition for enzyme treatment of mycelium was incubation at 37 °C for 15 min followed by 34 °C for 105 min. The amount of protoplasts produced was positively correlated with growth rate and negatively correlated with mycelial density. Under favorable conditions, R. solani mycelia released 1.68 × 106 protoplasts/mL that is comparable with that produced with Novozym 234. Among various media tested, the best solid medium for protoplast regeneration was 1% V-8 juice agar, while the best liquid medium was 10% potato dextrose broth.